Pyrimidine dimers are the most frequent and biologically significant of the lesions induced in DNA by far UV radiation. Left in place these lesions lead to mutagenesis, malignant transformation and cell death. The survival and genetic integrity of organisms depends upon the action of repair enzymes which remove these lesions from DNA. Individuals with the hereditary disorder xeroderma pigmentosum display a high incidence of skin cancer in areas exposed to UV light, reflecting deficient repair of pyrimidine dimers, and recent studies suggest that reduced DNA repair is a risk factor for development of UV-induced cancers in normal human populations. DNA photolyases repair pyrimidine dimers in DNA via a light- dependent reaction which restores the pyrimidine monomers in situ, and in addition stimulate the excision repair of dimers. This proposal describes experiments to elucidate how the Phr1 photolyase from Saccharomyces cerevisiae recognizes pyrimidine dimers in DNA, how the enzyme stimulates excision of these lesions, and how expression of the gene encoding this enzyme is regulated in response to DNA damage. Phr1 consists of a 66 kDa apoenzyme, encoded by the PHR1 gene, and two chromophores, and has been purified to homogeneity. Site directed mutagenesis will be used to alter conserved residues on the enzyme; the roles of these residues in substrate recognition will be assessed through quantitation of the specific and nonspecific equilibrium binding constants, while the location of altered contacts with substrate will be determined using footprinting techniques. The role of Phr1-mediated distortion of the substrate in binding and stimulation of dark repair will be addressed by examining the electrophoretic mobility of Phr1-pyrimidine dimer complexes in defined substrates. Linker insertion mutagenesis will be used to produce PHR1 mutants defective in dark (excision) repair stimulation (PHR1-drs). Potential targets for stimulation will be sought by isolating and cloning extragenic suppressors of the PHR1-drs mutants; these suppressors are likely to encode components of the incision complex formed as the initial step in excision repair. The gene encoding Prp, a damage-responsive repressor of PHR1 transcription, will be cloned and sequenced and the effect of increased and decreased levels of PRP expression on the expression of PHR1 and other known damage-inducible genes from yeast will be assessed by UV-survival. The mechanisms regulating loss of Prp binding will be investigated by determining the physical fate of Prp, changes in the level of Prp transcription, and post- translational modification of Prp following DNA damage. Deletion analysis and site-directed mutagenesis will be used to identify additional damage- responsive sequences within the PHR1 5' regulatory region.